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=Trypsin-modified Elongation Factor Tu (EF-Tu)=

General Information
Trypsin-modified Elongation Factor thermo unstable (Tm-EF-Tu) is a prokaryotic transcription factor and GTPase, which regulates the movement of aminoacyl tRNA into a free site of a ribosome. This protein has been studied in Escherichia coli (E. coli) and shows modification at three regions of the native peptide sequence; Arg-44, Arg-58, and Lys-56. Tm-EF-Tu tends to form a complex with the antibiotic tetracycline The resulting complex, Tm-EF-Tu-MgGDP, is composed of three domains and can be found in two crystalline structures.

Structure
EF-Tu is a 12 chain structure in E. coli that tends to form a complex with the antibiotic tetracycline. Two different crystalline forms of the structure have been identified. In the first crystal form, P21, six copies of EF-Tu, GDP, tetracycline, and Mg2+ are arranged as dimers in an asymmetric manner. An alternative form, P43212, is a monomeric, asymmetric unit with tetracycline complexes within the structure.

The P21 structure forms a final complex consisting of six EF-Tu proteins, GDP, tetracycline, Mg2+, and 244 water molecules. The final complex of the P43212 crystal is composed of one protein copy, GDP, tetracycline, Mg2+, SO2, glyoxylic acid, 3 Na+ and 160 H2O molecules. In either case, the binding site for tetracycline is located in domain one, allowing it to interact with the major functional groups. Residues Tet O11 and Tet O12 of tetracycline co-ordinate Mg2+. There is also an interaction with the phosphate of the GDP and the residues Thr25 and Asp80. When bound, tetracycline replaces two well ordered water molecules from the EF-Tu structure.



There are three domains making up the EF-Tu molecule. Domain 1, visualized in green, is the guanine-nucleotide domain made from residues 8-40 and 59-204. Domain 2, purple, is composed on residues 205-298 and Domain 3 , yellow, of residues 299-393. The fragments 8-44 and 59-393 are associated through non-covalent interactions and retain their native conformation, with the exception of residues 40-44 and 260-263.

In domain 1 of EF-Tu, the protein-protein interactions involved four anti-parallel β-sheet hydrogen bonds between residues 64-66’ and 66-64’ (where the primes indicate non-crystallographically related residues). Hydrogen bonds are also formed between the side chain of Asn63 and Glu68 as well as the main chain residues Ile62 with His66 and Glu60.

In the second domain, residues 216,259,261,262, and 287 form two electrostatic interactions and six hydrogen bonds form with 216’, 261’, 262’, and 283’ to create a mobile loop from Arg262-Leu264. There are also stabilizing van der Waals interactions between the dimers. The tetracycline rings are nearly parallel to the dimer unit creating a hydrophobic environment. Each dimer of the Tm-EF-Tu-MgGDP complex is related to another pair by a pseudo-twofold axis with minimal intermolecular interactions. Conformations of the protein complexed with tetracycline do not appear to differ from those which are non-complexed, suggesting little effect on the proteins structure.

Residues 40-44 and 260-263 are disordered when EF-Tu-MgGDP undergoes trypsin modification. In addition, residues 1-7 and 45-58 are removed, the latter of which makes up the Switch I loop. The Switch I loop conformation is regulated by the binding of MgGDP and MgGTP. The presence of tetracycline may affect the conversion rate between the EF-Tu-MgGTP and EF-Tu-MgGDP conformations, affecting the interaction with the ribosome. In this way, tetracycline may act to inhibit protein synthesis by preventing the binding of aminoacyl-tRNA to the A site of the ribosome.

Function
EF-Tu binds an aminoacylated tRNA molecule in the cytoplasm and allows entry into the ribosome. EF-Tu binds a cavity between the 30S and 50S ribosomal subunits, while the tRNA anticodon associates with the mRNA codon in the A site of the ribosome. If the pairing is incorrect, the tRNA will likely leave the ribosome. However, if a correct pairing has occurred, EF-Tu hydrolyzes guanosine triphosphate (GTP) to the diphosphate form (GDP). This hydrolysis results in a change in conformation, releasing the tRNA into the ribosome. The dissociation allows the tRNA molecule to then completely interact with the A site of the ribosome. Then the amino acid on the tRNA can be transferred to the growing peptide chain via covalent bond formation. The GDP-bound EF-Tu molecule can then interact with EF-Ts, which exchanges the GDP for a new GTP and prepares EF-Tu to interact with another charged amino acid, once EF-G has acted for translocation along the mRNA sequence. In this way, EF-Tu is a vital component in the process of lengthening a peptide during protein synthesis.

EF-Tu has also been shown to play a role in the inhibition of tetracycline during protein synthesis in many different organisms. EF-Tu may be a target protein of tetracycline, although this idea has commonly been dismissed because the ribosome may be inhibited in the presence of the antibiotic. Now however, it has been demonstated that Tm-EF-Tu-MgGDP is bound in a complex during crystallization, binding the the GTPase active site. This information could be useful for developing mechanisms of counteracting resistance to this particular antibiotic.